Dysfunction of glutamatergic neurotransmission is implicated in many neuropsychiatric disorders, including schizophrenia, epilepsy and autism. Although postsynaptic receptors have received the most attention, presynaptic mechanisms controlling glutamate release are also promising therapeutic targets, but have been less amenable to study. Glutamate release by synaptic vesicle exocytosis depends on glutamate packaging and recycling mediated by vesicular glutamate transporters (VGLUTs). VGLUT1 and 2 isoforms exhibit complementary expression in adult brain that distinguishes cortical (VGLUT1) and subcortical (VGLUT2) connections. Using genetically encoded optical reporters of glutamate transmission, VGLUT1 and 2-pHluorins, we have characterized the isoform-specific sorting signals and protein interactions that mediate differences in VGLUT1 and 2 trafficking. The involvement of proteins previously not associated with synaptic vesicle proteins may suggest novel mechanisms for vesicle recycling. Presynaptic signaling networks upstream of isoform- specific VGLUT trafficking present an opportunity to differentially modulate glutamate release in discrete brain pathways, and identify novel therapeutic targets to normalize brain circuits in neuropsychiatric disease. These mechanisms may also differentially depend on neuronal firing rate, offering the possibility of dampening excess activity while allowing normal physiological transmission to proceed. The long-term goal of the proposed research is to understand how membrane trafficking of individual vesicular proteins influences the protein composition of synaptic vesicles, the maintenance of synaptic vesicle pools, and the release of transmitter by specific circuits. The strategy of this proposal is to study signaling pathways upstream from isoform-specific VGLUT synaptic vesicle recycling. The specific aims of this proposal are designed to study the regulation of trafficking of vesicular glutamate transporters by 1) characterizing the modulation of VGLUT1 recycling by ubiquitin ligase interactions, 2) characterizing modulation of VGLUT2 recycling by inositol hexakisphosphate kinases, and 3) characterizing how isoform-specific trafficking changes over synapse development. As key mediators of synaptic transmission, these vesicular proteins and the factors that modulate their expression, localization and activity can dramatically influence neurotransmitter release, making them promising therapeutic targets. Regulation of neurotransmitter release may be an important approach to therapeutic intervention. The molecular machinery offers new targets for the development of better treatments for neuropsychiatric disorders.